Monoclonal antibodies have in recent years become successful therapeutic molecules, in particular for the treatment of cancer and autoimmune diseases. Effector functions mediated by the Fc region of antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are often important mechanisms for the clinical efficacy of monoclonal antibodies.
Antibody-induced CDC is mediated through the proteins of the classical complement cascade. This cascade is triggered by binding of the complement protein C1q to the antibody. C1q is composed of a bundle of six heterotrimeric subunits having globular heads and collagen-like tails. Binding of C1q to the Fc region of an antibody is known to involve the CH2 region of the antibody (Duncan and Winter (1988) Nature 332:738). Furthermore, sugar moieties on the Fc region are known to influence the binding of C1q (Raju (2008) Curr Opin Immunol 20:471).
Monoclonal antibodies are complex molecules that can undergo many types of enzymatic and non-enzymatic post-translational modifications, including formation of disulfide bonds, glycosylation, glycation, N-terminal glutamine cyclization, C-terminal lysine processing (removal), deamidation, isomerization, oxidation and peptide bond cleavage (for a review of these modifications, see by Liu et al. (2008) J Pharm Sci 97: 2426).
Removal of C-terminal lysines by carboxypeptidases from the heavy chain is a commonly observed antibody modification, both upon recombinant expression of antibodies in mammalian cells, as well as in vivo in human serum (Cai et al. (2010) Biotechnol. Bioeng. September 9). Removal is often partial, resulting in a mixed population of antibodies with zero (K0), one (K1) or two (K2) C-terminal lysines (i.e., in the case of K2, one C-terminal lysine in each heavy chain of the antibody). In particular, B cell hybridomas produce mixtures of K0, K1 and K2 molecules (Dick et al. (2008) Biotech. Bioeng. 100:1132).
WO 2009027471 describes that deletion of the codon for the C-terminal lysine of a heavy chain of an antibody can result in higher antibody titers upon expression in Chinese Hamster Ovary (CHO) cells.
WO 2008006554 describes that an antibody preparation produced in Δxyl-t/Δfuc-t moss cells comprises an N-glycan structure free of fucose and xylose and lacks C-terminal lysine residues. The ability of this preparation to mediate ADCC was less inhibited by serum than a preparation of the same antibody produced in murine Sp2/0 cells. Complement-mediated lytic activity, on the other hand, was reduced. Removal of the C-terminal lysine residue was proposed to increase ADCC, although the effect of this modification alone was not tested.
The general belief in the art has been that C-terminal lysines have little or no effect on antibody function, see e.g. the literature review by Cai et al. (2010) supra, which concluded that no activity had been attributed to C-terminal lysines, or Harris (2005) Dev Biol (Basel) 122:127 which stated that the presence or absence of heavy chain Lys residues had no effect on antigen binding, and was not likely to influence Fc effector functions, clearance or any other biological property.
Antes et al. (2007) J. Chromatogr. B 852:250 described testing the effect of C-terminal lysines on CDC. Antibody preparations were generated in Sp2/0 cells containing a significant subpopulation of C-terminal lysine-containing antibody molecules. Proteolytic removal of the C-terminal lysines had no effect on the ability of these antibody preparations to mediate CDC. The authors concluded, e.g., that both antibody variants—clipped and unclipped—elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.